Published in "Botanical Journal of the Linnean Society 192(4): 656–674, 2020" which should be cited to refer to this work. Systematics of Vriesea (Bromeliaceae): phylogenetic relationships based on nuclear gene and partial plastome sequences TALITA MOTA MACHADO1*, , ORIANE LOISEAU2, MARGOT PARIS3, ANNA WEIGAND4, LEONARDO M. VERSIEUX5, JOÃO RENATO STEHMANN1, CHRISTIAN LEXER6 and NICOLAS SALAMIN2, 1Departamento de Botânica, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Vegetal, Universidade Federal de Minas Gerais, Belo Horizonte, CEP 31270–901, MG, Brazil 2Department of Computational Biology, University of Lausanne, 1015 Lausanne, Switzerland 3Department of Biology, Unit Ecology and Evolution, University of Fribourg, Fribourg, Switzerland 4Department of Systematic and Evolutionary Botany, University of Zurich, 8008, Zurich, Switzerland 5Laboratório de Botânica Sistemática, Departamento de Botânica e Zoologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, CEP 59078–970, RN, Brazil 6Department of Botany and Biodiversity Research, Faculty of Life Sciences, University of Vienna, Rennweg 14, 1030 Vienna, Austria Vriesea is the second largest genus in Tillandsioideae, the most diverse subfamily of Bromeliaceae. Although recent studies focusing on Tillandsioideae have improved the systematics of Vriesea, no consensus has been reached regarding the circumscription of the genus. Here, we present a phylogenetic analysis of core Tillandsioideae using the nuclear gene phyC and plastid data obtained from genome skimming. We investigate evolutionary relationships at the intergeneric level in Vrieseeae and at the intrageneric level in Vriesea s.s. We sampled a comprehensive dataset, including 11 genera of Tillandsioideae and nearly 50% of all known Vriesea spp. Using a genome skimming approach, we obtained a 78 483-bp plastome alignment containing 35 complete and 55 partial protein-coding genes. Phylogenetic trees were reconstructed using maximum-likelihood based on three datasets: (1) the 78 483 bp plastome alignment; (2) the nuclear gene phyC and (3) a concatenated alignment of 18 subselected plastid genes + phyC. Additionally, a Bayesian inference was performed on the second and third datasets. These analyses revealed that Vriesea s.s. forms a well-supported clade encompassing most of the species of the genus. However, our results also identified several remaining issues in the systematics of Vriesea, including a few species nested in Tillandsia and http://doc.rero.ch Stigmatodon. Finally, we recognize some putative groups within Vriesea s.s., which we discuss in the light of their morphological and ecological characteristics. ADDITIONAL KEYWORDS: Atlantic Forest – chloroplast – epiphytes – genome skimming – monocotyledons – Neotropics – next-generation sequencing – Tillandsioideae. INTRODUCTION family has been suggested to be the result of rapid speciation and adaptive radiations mainly triggered by Bromeliaceae are a large, nearly entirely Neotropical the evolution of key innovations that enabled species plant family with a wide geographical distribution to colonize new adaptive zones (Givnish et al., 2011, ranging from the southern United States to northern 2014; Silvestro, Zizka & Schulte, 2014). Morphological Patagonia in Argentina; a single species occurs in features, such as tank-forming leaves, epiphytism, Africa (Smith & Downs, 1974). Diversification in the water- and nutrient-absorbing leaf trichomes and CAM photosynthesis, have allowed bromeliads to *Corresponding author. E-mail: [email protected] colonize highly heterogeneous environments (Benzing, 1 2000; Silvestro et al., 2014). Consequently, bromeliads Butcher & Gouda, cont. updated; Palma-Silva et al., occur from mesophytic forests to xerophytic rock 2016; Schütz et al., 2016). outcrops, from sea level (e.g. restingas in Atlantic Vriesea, the fourth largest genus in Bromeliaceae Forest) to the top of mountains in the Andes and the (Gouda et al., cont. updated), highlights well the Brazilian Shield (Gilmartin, 1973; Krömer, Kessler & problems faced by current phylogenetic studies. In Herzog, 2006; BFG, 2015, 2018). The wide variation the genus, 88% of the species in the genus occur in in morphological characters and the low rates of Brazil, and 84% of all Vriesea spp. are endemic to the molecular evolution observed in Bromeliaceae (Smith country (BFG, 2015, 2018). They are distributed from & Donoghue, 2008; Maia et al., 2012) make this family xeric to mesic environments (Versieux & Wendt, 2007; of c. 3587 species in 75 genera (Butcher & Gouda, cont. Versieux, 2008; Costa, Gomes-da-Silva & Wanderley, updated) a challenging group for taxonomists. Because 2014; Machado, Forzza & Stehmann, 2016) and are a of these characteristics, the delimitation of species, or conspicuous element of the Atlantic Forest (Martinelli sometimes even genera, has proved difficult (Palma- et al., 2008). Some species reach a western inland Silva et al., 2016). distribution, growing as epiphytic, terrestrial or Based on molecular phylogenetic analyses, eight rupicolous plants on inselbergs and in rocky savanna- subfamilies of Bromeliaceae, the circumscription of like habitats (Versieux & Wendt, 2007; Versieux et al., which was usually based on only a few plastid markers, 2008; Costa et al., 2014). The geographical distribution are currently recognized in the literature (Terry, Brown of species varies from wide ranges covering different & Olmstead, 1997a, b; Horres et al., 2000; Crayn, habitats to microendemics that are particularly Winter & Smith, 2004; Barfuss et al., 2005; Givnish frequent in mountainous environments (Versieux & et al., 2007). In the last decades, many phylogenetic Wendt, 2007; Costa et al., 2014; Machado, Forzza & studies have improved our understanding of the Stehmann, 2016). Due to the restricted distributions of evolutionary history of these subfamilies, but species many Vriesea spp. and the continuous loss of habitat, representation has been highly heterogeneous among the genus is the second most endangered in Brazil studies (Escobedo-Sarti et al., 2013; Palma-Silva et al., when considering the absolute number of endangered 2016). For instance, among the subfamilies with the plant species (Martinelli et al., 2013). The wide climatic highest number of published phylogenetic studies tolerance of Vriesea is coupled with morphological (Bromelioideae and Tillandsioideae), Tillandsioideae variation (Costa et al., 2014; Costa, Gomes-da-Silva & are also the subfamily with the most scattered and Wanderley, 2015). Phenotypic variability occurs also at least connected sampling because most publications the intraspecific level, making the delimitation of taxa analysed only small clades (Escobedo-Sarti et al., complicated, and leading to the recognition of several 2013). As a result of the low rates of substitution in species complexes (Almeida et al., 2009; Gomes-da- plastid regions, the tree topologies are often poorly Silva & Costa, 2011; Versieux, 2011; Neves et al., 2018). resolved not helping in the circumscription of some The polyphyletic condition of Vriesea and the genera in Bromeliaceae (Smith & Donoghue, 2008; difficulty in recognizing unique morphological Maia et al., 2012; Palma-Silva et al., 2016); non-natural characters to circumscribe the genus have been known genera are frequently reported in Bromeliaceae for over two decades (Terry et al., 1997a; Barfuss (Barfuss et al., 2005; Evans et al., 2015; Schütz et al., et al., 2005; Givnish et al., 2011; Gomes-da-Silva et al., 2016; Maciel et al., 2018) and only 30% of the currently 2012; Costa et al., 2015; Gomes-da-Silva & Souza- http://doc.rero.ch recognized genera are monophyletic (Escobedo-Sarti Chies, 2017). Recent studies using morphological et al., 2013). To overcome these problems, recent characters and molecular markers have elucidated studies have increased the amount of analysed data the intergeneric relations in Tillandsioideae and either by increasing the number of molecular markers helped to improve the circumscription of Vriesea and their phylogenetic informativeness (e.g. more (Barfuss et al., 2016; Gomes-da-Silva & Souza-Chies, quickly evolving nuclear regions or even AFLP) or 2017). However, conflicting results are found in the increasing the numbers of terminals (Krapp et al., literature, potentially due to the low species sampling 2014; Heller et al., 2015; Barfuss et al., 2016; Pinangé of Vriesea, which remains poorly representative of the et al., 2016; Schütz et al., 2016; Goetze et al., 2017; whole genus. To achieve a robust classification, the Gomes-da-Silva & Souza-Chies, 2017; Leme et al., last taxonomic revision of Tillandsioideae proposed a 2017; Maciel et al., 2018; Matuszak-Renger et al., series of rearrangements dividing large polyphyletic 2018). Nevertheless, large and complex to delimit genera into small, monophyletic, well-circumscribed genera such as Tillandsia L. (741 species), Pitcairnia morphological groups (Barfuss et al., 2016). Based L’Heritier (408 species), Vriesea Lindl. (226 species), on the stigma morphology, Vriesea s.l. was split into Puya Molina (226 species) and Guzmania Ruiz & Pav. five new genera (Goudaea W.Till & Barfuss, Jagrantia (218 species) remain vastly under-sampled (Gouda, Barfuss & W.Till, Lutheria Barfuss & W.Till, Zizkaea 2 W.Till & Barfuss, Stigmatodon Leme, G.K.Br. & morphological and geographical variation of the Barfuss) and Vriesea s.s. (the Brazilian lineage) was species and type localities.